Project Summary Several genera of maternally inherited symbiotic bacteria affect the reproductive biology of their host species by favoring infected female over male offspring. Wolbachia are the archetypes of this adaptive strategy and exist globally in an estimated 40% of all arthropod species and many filarial nematodes, making them one of the most prevalent bacterial infections on the planet. In arthropods, Wolbachia can selectively kill sons of infected females in a process dubbed male killing. This form of sex-specific virulence enhances the spread of the bacteria by increasing the fitness of transmitting females through reduced competition with their dead brothers for limited resources. This type of bacterial drive occurs in many species of the three largest insect orders including Coleoptera, Diptera, and Lepidoptera, and arachnids. It can also spur host evolutionary responses including selection for male mate choice or host resistance as males become rare in populations experiencing high levels of male killing. Regarding vector control, male killing is a proposed strategy to eliminate or suppress host targets. Population models specify that male killing bacteria can greatly increase the efficiency of existing techniques. However, despite almost a century of research on male killing and its relevance to ecology, evolution, and vector control, a genetic basis remains enigmatic and one of the field's most central challenges to solve. We recently identified a candidate gene, hereafter known as WO Male Killing (wmk), from the prophage WO region of the wRec Wolbachia strain of Drosophila recens that can cause male killing. When wmk is transgenically expressed in uninfected D. melanogaster, it induces a partial, male killing-like phenotype by decreasing the number of surviving male offspring and the embryonic hatch rate compared to controls. Additionally, cytological defects caused by male killing Wolbachia are enriched in male wmk embryos versus controls. There are also significantly more late stage inviable wmk embryos compared to controls, and that number matches the decrease in surviving males. Finally, as wmk is a putative transcriptional regulator, RNA-seq in wmk male versus control embryos revealed altered transcription of many host genes. As wmk represents a potential breakthrough in the search for a bacterial gene that causes male killing, we propose the first in-depth examination of a putative Wolbachia male killing gene and its interactions with the host. This proposal will therefore test the central hypothesis that wmk kills males by altering host transcription via interactions with the host DNA. Proposed experiments will be to (i) perform ChIP-seq to confirm DNA binding and identify host target genes, and (ii) determine if Wmk depends on host candidate genes (identified in RNA-seq and ChIP-seq) to kill males by expressing wmk in mutant, knockout, or RNAi Drosophila strains for each candidate. This will allow an identification and functional analysis of Wmk- DNA interactions. Examinations to date have yet to reveal the bacterial genetic basis of Wolbachia male killing. If successful, this research will identify the mechanistic basis of Wolbachia male killing inform the future use of transgenic or paratransgenic strategies to curb the transmission of insect-borne diseases and spread of pests.